Our laboratory has a longstanding interest in the molecular pathogenesis of bone marrow failure, a condition characterized by the inability of the bone marrow to produce sufficient blood cells. Bone marrow failure may affect all three major blood cell lineages (erythrocytes, leukocytes and platelets); in other cases, one lineage is disproportionately affected. Here we propose to newly investigate the mechanisms that lead to Diamond Blackfan Anemia (DBA) a rare bone marrow failure syndrome presenting mainly in early infancy. DBA is associated with the decreased production or absence of erythroid precursors in the bone marrow, variable congenital anomalies, and a predisposition to malignancy. DBA is a genetically heterogeneous disease. Mutations in the ribosomal protein RPS19 have been identified in about 25% of cases with DBA. The pathway that leads from a mutation in RPS19, a ubiquitously expressed protein, to a specific defect in terminal differentiation of red cells is not understood. In this pilot and feasibility project we will test the hypothesis that the underlying mechanism responsible for the clinical manifestations in patients with DBA is a defect in ribosome biogenesis. We propose 1) that RPS19 mutations cause a defect in pre-ribosomal RNA processing and ribosome biogenesis that, although present in all growing cells from patients with DBA, is detrimental for the rapidly dividing and synthetically highly active early erythroid progenitor cell and 2) that the identical pathway is affected in patients who have DBA but no RPS19 mutation. To test this hypothesis we will investigate rRNA processing and ribosome biogenesis in lymphoblastoid cell lines established from 1) patients with DBA due to an RPS19 mutation, 2) normal individuals and 3) DBA patients who do not have an RPS19 mutation. By investigating rRNA precursors we will test whether rRNA processing proceeds in a normal fashion, whether observed abnormalities are specific for the RPS19 mutation, or whether similar alterations are found in individuals with DBA but no RPS19 mutation. We will investigate the dynamics of ribosome biogenesis by analysis of the profile of ribosomal subunits, intact ribosomes and polysomes. Using a proteomic approach we will specifically test whether the mutant RPS19 protein participates in the biogenesis of ribosomes and is part of translationally active polysomes. The results will show whether cells from patients with DBA have a defect in rRNA processing and ribosome biogenesis which will enable us to focus on these pathways in our future research. Knowledge of the course of ribosome biogenesis in normal and DBA hematopoietic cells might lead to the development of exciting new tools that may be used a) for more specific and sensitive diagnosis of DBA, b) for the identification and characterization of the molecular defect(s) in individuals with DBA but no RPS19 mutation and finally, c) for the identification of new targets for the development of specific and novel reagents that may be tested for the treatment of individuals with DBA.